Mammalian cells must integrate, and respond to, a myriad of signals from their microenvironment. Many of these signals are sensed by receptors expressed on the surface of the responding cell. Two critical classes of cell surface receptors include those known as "receptor tyrosine kinases" and those classified as "integrins". Receptor tyrosine kinases recognize and respond to peptide growth factors such as insulin, platelet-derived growth factor and nerve growth factor (Ullrich and Schlessinger, 1990), while the integrins most often mediate binding and attachment to components of the extracellular matrix such as collagen, fibronectin, and vitronectin (Clark and Brugge, 1995). There is increasing evidence that receptor tyrosine kinases and integrins act in coordinated fashion to modulate cellular responses involving adhesion, spreading, locomotion, proliferation, survival and differentiation state (Clark and Brugge, 1995).
Receptor tyrosine kinases are thus named because of the tyrosine kinase domain found in the cytoplasmic portion of these receptors (Ullrich and Schlessinger, 1990). Ligand binding to the receptor ectodomain results in activation of the tyrosine kinase domain, which in turn leads to recruitment and activation of a variety of downstream signaling molecules. A number of receptor-like tyrosine kinases have been molecularly cloned based on the homologies shared by the tyrosine kinase domains of all receptors in this class (e.g., Lai and Lemke, 1991). Although presumed to have ligands, these receptor-like proteins are termed "orphans" until their ligands are indeed identified. A variety of approaches have led to the identification of ligands for previously orphan receptors. For example, the ephrins have been identified as the ligands for the EPH family of receptors (Bartley et al., 1994; Beckmann et al., 1994; Davis et al., 1994; Cheng and Flanagan, 1994), Protein S and Gas6 have been identified as ligands for the Tyro3/Sky/rse/brt/tf and Axl/Ark/UFO receptors (Stitt et al., 1995; Varnum et al., 1995), agrin has been identified as the ligand for MuSK (Glass et al., 1996), glial-derived neurotrophic factor has been identified as the ligand for the Ret receptor (Jing et al., 1996; Treanor et al., 1996), and the angiopoietins have been identified as the ligands for the Tie receptors (Davis et al., 1996; Maisonpierre et al., 1997).
Among the few remaining orphan receptor-like tyrosine kinases are two close relatives which are distinguished by a strucutral domain in their extracellular portions that has not been found in other receptor tyrosine kinases, but was instead first noted in the discoidin I protein of the slime mold Dictyostelium discoideum (Poole et al., 1981) and thus termed the discoidin I domain. Discoidin I domains have more recently been noted to be homologous to the constant regions of blood coagulation Factors V and VIII (Wood et al., 1984; Jenny et al., 1987) and to a neural recognition molecule termed A5 identified in Xenopus laevis (Takagi et al., 1987). The two closely related receptor-like tyrosine kinases which contain discoidin I domains have been cloned by several groups and given several different names. We will refer to these receptor-like tyrosine kinases as Discoidin Domain Receptor 1 (DDR1) for the receptor previously termed DDR (Johnson et al., 1993), NEP (Zerlin et al., 1993), Ptk-3 (Sanchez et al., 1994), Cak (Perez et al., 1994), trkE (DiMarco et al., 1993) and MCK-10 (Alves et al., 1995), and Discoidin Domain Receptor 2 (DDR2) for the receptor previously termed Tyro10 (Lai and Lemke, 1991; Lai and Lemke, 1994), TKT (Karn et al., 1993) and CCK-2 (Alves et al., 1995). Previous studies have found that DDR1 and DDR2 are quite widely but differentially expressed during development and in the adult.
Regions of homology in the Trks as well as other RTKs, in combination with the use of PCR technology, has rapidly enabled the cloning of an abundant number of novel protein tyrosine kinases, wherein the cognate ligand has yet to be discovered (hence, such receptors are termed "orphan" receptors). For example, Lai and Lemke (Neuron 6: 691-704 (1991)) identified thirteen novel kinases, designated Tyro-1 through Tyro-13, with several bearing similarity to other known RTKs. Structural comparison indicates that the tyrosine kinase domain of Tyro-10 (DDR-2) is most closely related to the equivalent domains of the Trks. (Lai & Lemke, 1994, Oncogene 9: 877-883). Similarly, Zerlin et al. (Oncogene 8: 2731-2739 (1993)) report the molecular cloning of a cDNA encoding a novel receptor protein tyrosine kinase designated NEP (DDR-1), that is highly expressed in proliferating neuroepithelia. The authors suggested that one function of NEP (DDR-1) kinase is to signal proliferation of neuroepithelial cells in response to an as yet unknown ligand.
Despite the lack of known cognate ligands, knowledge of the tissues in which such orphan receptors are expressed provides insight into the regulation of the growth, proliferation and regeneration of cells in the tissues. Because RTKs appear to mediate a number of important functions associated with development and maintenance, identification of their cognate ligands will inevitably play a crucial role in characterizing these functions.
Ligand-receptor assays are generally useful for the in vitro determination of the presence and concentration of ligands in body fluids, food products, animal fluids, and environmental samples. For example, using such assays to determine the presence and concentration of specific hormones, proteins, therapeutic drugs, and toxic drugs in human blood or urine has significantly improved medical diagnosis.
Ligand-receptor assays rely on the binding of ligands to receptors to determine the presence and/or concentration of ligands in a sample. Ligand-receptor assays can be described as either competitive or non-competitive. Non-competitive assays generally utilize receptors in substantial excess over the concentration of ligand to be determined in the assay. Sandwich assays, in which the ligand is detected by binding to two receptors, one receptor labeled to permit detection and a second receptor frequently bound to a solid phase to facilitate separation from unbound reagents, such as unbound labeled first receptor, are examples of non-competitive assays.
Competitive assays generally utilize ligand from the sample, a ligand analogue labeled to permit detection, and the competition of these species for a limited number of binding sites provided by the ligand receptor. Those skilled in the art will appreciate that many variations of this basic competitive situation have been previously described. Examples of ligands which are commonly measured by competitive ligand-receptor assays include haptens, hormones and proteins. Antibodies or receptorbodies that can bind these classes of ligands are frequently used in these assays as ligand receptors.
Competitive ligand-receptor assays can be further described as being either homogeneous or heterogeneous. In homogeneous assays, all of the reactants participating in the competition are mixed together and the quantity of ligand is determined by its effect on the extent of binding between ligand receptor and labeled ligand analogue. The signal observed is modulated by the extent of this binding and can be related to the amount of ligand in the sample. U.S. Pat. No. 3,817,837 describes such a homogeneous, competitive immunoassay in which the labeled ligand analogue is a ligand-enzyme conjugate and the ligand receptor is an antibody capable of binding to either the ligand or the ligand analogue. The binding of the antibody to the ligand-enzyme conjugate decreases the activity of the enzyme relative to the activity observed when the enzyme is in the unbound state. Due to competition between unbound ligand and ligand-enzyme conjugate for antibody binding sites, as the ligand concentration increases the amount of unbound ligand-enzyme conjugate increases and thereby increases the observed signal. The product of the enzyme reaction may then be measured using a spectrophotometer.
In general, homogeneous assay systems require both an instrument to read the result and calibration of the observed signal by separate tests with samples containing known concentrations of ligand. The development of homogeneous assays has dominated competitive assay research and has resulted in several commercially available systems.
Heterogeneous, competitive ligand-receptor assays require a separation of bound labeled ligand or receptor from the free labeled ligand or receptor and a measurement of either the bound or the free fraction. Methods for performing such assays are described in U.S. Pat. Nos. 3,654,090, 4,298,685, and 4,506,009. U.S. Pat. Nos. 4,125,372, 4,200,690, 4,246,339, 4,366,241, 4,446,232, 4,477,576, 4,496,654, 4,632,901, 4,727,019, and 4,740,468 describe devices and methods for ligand-receptor assays that develop colored responses for visual interpretation of the results.
In the case of a competitive immunoassay, a labelled antigen reagent is bound to a limited and known quantity of antibody reagent. After that reaction reaches equilibrium, the antigen to be detected is added to the mixture and competes with the labelled antigen for the limited number of antibody binding sites. The amount of labelled antigen reagent displaced, if any, in this second reaction indicates the quantity of the antigen to be detected present in the fluid sample.
Because competitive assays generally result in non-linear response functions, several calibration points are required for such assays in order to determine the response over the assay range. In order to simplify the calibration process, two extreme approaches have evolved. One approach is not to reduce the number of calibrators or replicates needed to determine the response but to reduce the frequency of such calibration. Such assays rely upon instruments to perform the assay and to control variables that affect the assay response so that calibration is infrequent or is performed by the manufacturer and does not need to be performed by the user of the assay. The second approach is to not use an instrument and to provide a simplified means of calibration so that no additional tests are needed to calibrate the assay response.
The method of U.S. Pat. No. 4,540,659 provides an assay for the quantitation of ligand in samples where predetermined ratios of responses at a calibration surface and a measurement surface are related to the concentration of the ligand.
Another approach, a non-competitive immunochromatographic assay, is described in U.S. Pat. Nos. 4,168,146 and 4,435,504. This assay provides a method for quantitatively determining the presence of a single analyte in a sample in a visually interpreted immunoassay. U.S. Pat. No. 5,089,391 describes a method for performing competitive ligand-receptor assays so as to be able to semiquantitatively or quantitatively determine the concentration of the ligand.